Immunopotentiatory agents and physiologically acceptable salts thereof

ABSTRACT

The present invention relates to the use of 4-(2-formyl-3-hydroxyphenoxymethyl) benzoic acid or physiologically acceptable salts thereof as an immunopotentiatory agent, compositions containing such a compound and their manufacture, combinations of such a compound with anti-tumour or anti-infective drugs and the use of such combinations in the prophylaxis or treatment of such diseases arising from tumours or infections.

This is a continuation of Ser. No. 08/462,115 filed on Jun. 5, 1995, nowabandoned which is a continuation of application Ser. No. 08/224,152filed on Apr. 7, 1994, now abandoned which is a continuation-in-part ofU.S. Ser. No. 08/112,849 filed Aug. 26, 1993 now abandoned.

The present invention relates to the use of4-(2-formyl-3-hydroxyphenoxymethyl) benzoic acid as animmunopotentiatory agent, compositions containing such a compound andtheir manufacture, combinations of such a compound with anti-tumour oranti-infective drugs and the use of such combinations in the prophylaxisor treatment of such diseases arising from tumours or infections.

European Patent No: 54924 discloses4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid (the compound of formula(I): ##STR1## its synthesis and its properties as a "left-shifter" ofthe oxygen-dissociation curve. European Patent No. 54924 discloses thatthe compound of formula (I) increases the affinity of haemoglobin foroxygen causing transient hypoxia in normal and tumour tissue, and can beused in the radiosensitization of tumours. All references identifiedherein above or in the following are hereby incorporated by referencethereto. The generic name for the compound of formula (I) is tucaresol.

The principal protective function of the immune system relates toresistance to invasion by pathogens, including viruses, rickettsia,mycoplasma, bacteria, fungi and parasites of all types. Thus,improvement of immune response, particularly when depressed, improvesresistance to infection or infestation by any of the above pathogens.

A second protective function of the immune system is to resistengraftment of foreign tissue, either natural or in fetal-maternalrelationship; or unnatural as performed by the transplant physician.

A third protective function of the immune system is thought to beresistance to malignant cell development as in cancer. The use ofimmunopotentiators in cancer is logical to enhance tumour rejection andto inhibit tumour recurrences following other forms of therapy.

A fourth protective function involves maintaining non-reactivity to selfby positive suppressor mechanisms. In auto-immune and related disorders,immune reactivity directed at self antigens or exaggerated, elevatedresponses are apparent which are self destructive.

Each of the protective functions of the immune system can be modified bynonspecific therapy with immunopotentiators alone or in combination withother agents employed to improve resistance or to kill the invadingpathogen. In addition, specific resistance can be augmented by use ofimmunopotentiators in conjunction with some form of antigen as in avaccine employing, for example, a virus, tumour cells, etc. This use canbe to induce either specific immunity or tolerance. The latter isexemplified by use with an antigen in allergy or an auto-immune disease.Use of immunopotentiators may be either therapeutic or prophylactic; thelatter is used particularly in the elderly, where infection,auto-immunity, and cancer are more common. The timing and route ofadministration are critical in determining whether a positive ornegative response results. Any agent capable of augmenting an immuneresponse may inhibit it depending on timing and dose; thus, undercertain circumstances an immunopotentiator could be used as animmunosuppressive agent for use in allergy, auto-immunity andtransplantation.

By immunopotentiator is meant an agent which is capable of restoring adepressed immune function, or enhancing normal immune function, or both.However because of certain elements of the immune system animmunopotentiator can also have immunosuppressive effects.

Immune responses are orchestrated by T-lymphocytes whose stereo-specificreceptors are triggered by antigenic fragments bound to specialisedmolecules (MHC) on the surface of antigen presenting cells. In additionto the specific interaction between a T-cell receptor and antigen-MHCcomplex, T-cells require costimulatory signals which are provided byantigen-presenting cells. The interaction between antigen-presentingcells and T-cells involves accessory adhesion molecules some of whichare linked to the T-cell receptor:antigen-MHC interaction and otherswhich are separate from this interaction. Some of these adhesionmolecules provide costimulatory signals. In addition, in vitro studieshave established that transient covalent, chemical reactions betweencell surface ligands are essential in the antigen-specific activation ofT-cells Rhodes, J. (1989), J.Immunol. 143:1482; Gao, X. M. & Rhodes, J.(1990), J.Immunol. 144-2883; Rhodes, J. (1990), Immunol. 145:463). Thesetake the form of a carbonyl-amino condensation (Schiff base formation)between groups on APC and T-cell surface.

It has now been found that the compound of formula (I) orphysiologically acceptable salts thereof, surprisingly acts as animmunopotentiator.

In the salts of the compound of formula (I) the biological activityresides in ether (anion) moiety and the identity of the cation is ofless importance although for use in medicine it is preferablypharmacologically acceptable to the recipitent. Suitable salts includeammonium salts, alkali metal salts such as sodium and potasium salts,and salts formed with organic bases.

Therefore, the compound of the invention or physiologically acceptablesalts thereof may be used for the treatment of diseases where there is adefect in the immune system and/or an ineffective host defencemechanism, or to enhance activity of the immune system above normallevels.

The present invention provides for the use of4-(2-formyl-3-hydroxyphenoxymethyl) benzoic acid or physiologicallyacceptable salts thereof for the manufacture of a medicament for thepotentiation of an immune response.

By potentiation of an immune response is meant restoration of adepressed immune function, enhancement of a normal immune function, orboth. Immune function is defined as the development and expression ofhumoral (antibody-mediated) immunity, cellular (T-cell-mediated)immunity, or macrophage and granulocyte mediated resistance.

In this specification the term "immunodeficient patient" will be used todescribe patients with a deficient or defective immune system. Animmunodeficient patient can be characterised by means of a T-lymphocyteproliferation assay. Using this assay immunodeficient patients arecharacterised by a reduced ability of the T-cells to respond tostimulation by mitogens and recall antigens. An example of a mitogencommonly used in this assay is phytohaemagglutinin (PHA) and tetanustoxoid respectively.

In Adler et al., Cancer (1980) 45, 2062-2063 the immune function ofbreast cancer patients was evaluated by means of the T-lymphocyteproliferation assay using PHA. Quantitative estimation of the lymphocyteresponse to PHA was based on Stimulation Index (SI). In theaforementioned paper a SI value below 14 was defined to be below normaland thus these patients could be regarded as immunodeficient.Accordingly in this application we also consider that cancer patientswho have a SI value below 14 are immunodeficient.

There is a variety of circumstances in which the immune system may bedefective or deficient. Thus, for example immune system deficiency iscommon in immature or premature infants (neonates). It may also resultfrom suppression by certain drugs, which may be deliberate e.g. as aside-effect of cancer chemotherapy. Disordered growth of one or moreconstituent parts of the immune system, e.g. as in certain forms ofcancer, may also result in immunodeficiency. Immune deficiency mayfurthermore be caused by viral infections, including humanimmunodeficiency virus (HIV).

A further aspect of the present invention provides a method of treatingimmunodeficient patients, which comprises administering to a mammal(including human) an effective amount of the compound of formula (I), ora physiologically acceptable salt thereof. By an "effective amount" ismeant the amount of the compound of formula (I) which will restoreimmune function to normal levels, or increase immune fuction abovenormal levels in order to eliminate infection.

The compound of formula (I) or physiologically acceptable salts may beadministered for the treatment or prophylaxis of immunodeficient mammalsalone or in combination with other therapeutic agents, for example, withother antiviral agents, or with other anticancer agents.

According to a further aspect of the present invention provides for theuse of the compound of formula (I) or physiologically acceptable saltsthereof for the treatment and/or prophylaxis of acute and chronic viralinfections.

Examples of acute viral infections against which immunopotentiatorytherapy with the compound of formula (I) or physiologically acceptablesalts thereof may be used, preferably in conjunction with an antiviralagent, are:

Herpes viruses, influenza viruses, parainfluenza viruses, adenoviruses,coxsakie viruses, picorna viruses, rotaviruses, heptatitis A virus,mumps virus, rubella virus, measles virus, pox viruses, respiratorysyncytial viruses, papilloma viruses, and enteroviruses, arenavirus,rhinoviruses, poliovirus, Newcastle disease virus, rabies virus,arboviruses.

Examples of chronic viral infections against which immunopotentiatorytherapy with the compound of formula (I) or physiologically acceptablesalts thereof may be used are:

Persistent herpes virus infections, Epstein Barr virus infection,persistent rubella infections, papovirus infections, hepatitis virusinfections and human immunodeficiency virus infection.

The present invention is applicable to the treatment of viral hepatitisin all of its forms, five types now being recognised hepatitis A, B, C,D and E respectively.

Of the DNA viruses, those of the herpes group are the sources of themost common viral illnesses in man. The group includes herpes simplexvirus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV);Epstein-Barr virus (EBV) and human herpes virus 6 (HHV6). HSV 1 and HSV2 are some of the most common infectious agents of man. Most of theseviruses are able to persist in the host's neural cells; once infected,individuals are at risk of recurrent clinical manifestations ofinfection which can be both physically and psychologically distressing.

HSV infection is often characterised by extensive and debilitatinglesions of the skin, mouth and/or genitals. Primary infections may besubclinical although tend to be more severe than infections inindividuals previously exposed to the virus. Ocular infection by HSV canlead to keratitis or cataracts thereby endangering the host's sight.Infection in the newborn, in immunocompromised patents including AIDSpatients or penetration of the infection into the central nervoussystem, can prove fatal.

Varicella zoster (VZV) is a herpesvirus which causes chickenpox andshingles. Chickenpox is the primary disease produced in a host withoutimmunity and in young children is usually a mild illness characterisedby a vesicular rash and fever. Shingles or zoster is the recurrent formof the disease which occurs in adults who were previously infected withvaricella-zoster virus. The clinical manifestions of shingles arecharacterised by neuralgia and a vescicular skin rash that is unilateraland dermatomal in distribution. Spread of inflammation may lead toparalysis or convulsions. Coma can occur if the meninges becomesaffected. In immunodeficient patients VZV may disseminate causingserious or even fatal illness. VZV is of serious concern in patientsreceiving immunosuppressive drugs for transplant purposes or fortreatment of malignant neoplasia and is a serious complication of AIDSpatients due to their impaired immune system.

In common with other herpes viruses, infection with CMV leads to alifelong association of virus and host and following a primaryinfection, virus may be shed for a number of years. Congenital infectionfollowing infection of the mother during pregnancy may give rise toclinical effects such as death or gross disease (microcephaly,hepatosplenomegaly, jaundice, mental retardation), retinitis leading toblindness or, in less severe forms, failure to thrive, andsusceptibility to chest and ear infections. CMV infection in patientswho are immunocompromised for example as a result of malignancy,treatment with immunosuppressive drugs following transplantation orinfection with HIV may give rise to retinitis, pneumoitis,gastrointestinal disorders and neurological diseases CMV infection inAIDS patients is a predominant cause of morbidity in 50-80% of the adultpatient population, it is present in a latent form and can bere-activated in immunocompromised patients.

Epstein-Barr virus (EBV) causes infectious mononucleosis and hairyleukoplakis, and is also suggested as the causative agent of humancancer, such as nasopharyngeal cancer, immunoblastic lymphoma, Burkitt'slymphoma.

HBV is a viral pathogen of world-wide major importance. The virus isaetiologically associated with primary hepatocellular carcinoma and isthought to cause about 80% of the world's liver cancer. In the UnitedStates more than ten thousand people are hospitalised for HBV illnesseach year, and average of 250 die with fulminant disease. The UnitedStates currently contains an estimated pool of 500,000 to 1-millioninfectious carriers. Chronic active hepatitis generally develops in over25% of carriers, and often progresses to cirrhosis. Clinical effects ofinfection with HBV range from headache, fever, malaise, nausea,vomiting, anorexia and abdominal pains. Replication of the virus isusually controlled by the immune response, with a course of recoverylasting weeks or months in humans, but infection may be more severeleading to persistent chronic liver disease outlined above.

Of the RNA viruses, one group has assumed a particular importance thisis the retroviruses. Retroviruses form a sub-group of RNA viruses which,in order to replicate, must first `reverse transcribe` the RNA of theirgenome into DNA (`transcription` conventionally describes the synthesisof RNA from DNA). Once in the form of DNA, the viral genome may beincorporated into the host cell genome, allowing it to take advantage orthe host cell's transcription/translation machinery for the purposes ofreplication. Once incorporated, the viral DNA is virtuallyindistinguishable from the host's DNA and, in this state, the virus maypersist for the life of the cell.

In the case of immunosuppression resulting from HIV infection,prophylaxis may be required by those diagnosed as seropositive for HIVi.e. having antibodies to HIV, and those with PGL (progressivegeneralised lymphadenopathy) or ARC (AIDS-related complex) as well aspatients suffering from AIDS or patients suffering from AIDS-like immunedeficiencies where the HIV infection is not detectable and who alsorequire immunorestoration by means that are not specific to anyparticular virus.

The compounds according to the invention may be employed alone or incombination with other therapeutic agents for the treatment of the aboveinfections or conditions. Combination therapies according to the presentinvention comprise, the administration of at least one compound of theformula (I) or a physiologically functional derivative thereof and atleast one other pharmaceutically active ingredient. The activeingredient(s) and pharmacologically active agents may be administeredtogether or separately and, when administered separately this may occursimultaneously or sequentially in any order. The amounts of the activeingredient(s) and pharmacologically active agents(s) and the relativetimings of administration will be selected in order to achieve thedesired combined therapeutic effect. Preferably the combination therapyinvolves the administration of one compound of the formula (I) or aphysiologically functional derivative thereof and one of the agentsmentioned herein below.

Examples of such further therapeutic agents include agents that areeffective for the treatment of HIV infections or associated conditionssuch as 3'-azido-3'-deoxythymidine (zidovudine), other2',3'-dideoxynucleosides such as 2',3'-didoxycytidine,2',3'-dideoxyadenosine and 2',3'-didoxyinosine, carbovir; acyclicnucleosides (for example, acyclovir), 2',3'-didehydrothymidine, proteaseinhibitors such as N-tert-butyl-dechydro-2- -2(R)-hydroxy-4-phenyl-3(S)-N-2-quinolyl-carbonyl)-L-asparginyl!butyl!-(4aS,8aS)-isoquinoline-3(S)-carboxamide(Ro 31-8959), oxathiolan nucleoside analogues such ascis-1-(2-hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine orcis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluoro-cytosine,3'-deoxy-3'-fluorothymidine, 2',3'-dideox-5-ethynyl-3'-fluorouridine,5-chloro-2',3'-dideoxy-3' fluorouridine, Ribavirin, 9-4-hydroxy-2-(hydroxymethyl)but-1-yl!guanine (H2G), TAT inhibitors suchas 7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one (Ro5-3335), or7-chloro-1,3-dihydro-5-(1H-pyrrol-7-yl)-3H-1,4-benzodiazepin-2-amine(Ro24-7429) interferons such as α-interferon, renal excretion inhibitorssuch as probenecid, nucleoside transport inhibitors such asdipyridamole; pentoxifyline, NAcetylCysteine, Procysteine,α-trichosanthin, phosphonoformic acid, as well as immunodulators such asinterleukin II, granulocyte macrophage colony stimulating factors,erythropoetin, soluble CD₄ and genetically engineered derivativesthereof. Examples of such further therapeutic agents which are effectivefor the treatment of HBV infections include carbovir, oxathiolannucleoside analoges such ascis-1-(2-hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine orcis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl-5-fluoro-cytosine,2',3'-didoxy-5-ethynyl-3'-fluorouridine,5-chloro-2',3'-didoxy-3'-fluorouridine,1-(β-D-arabinofuranoysl)-5-propynyluracil, acyclovir and interferons,such as α interferon.

It has been reported (Hughes, W. T. (1987) Treatment and Prophylaxis ofPneumocystis carinii pneumonia, Parasitology Today 3(11) 332-335) thatat least 60% of patients with acquired immunodeficiency syndrome (AIDS)suffer from Pneumocystis carinii pneumonia.

Without treatment, Pneumocystis carinii pneumonia is almost always fatalin immunocompromised hosts. The most widely used treatments for thiscondition are trimethoprim-sulphamethoxazole (cotrimoxaole) andpentamidine. However, both of these treatments have been reported to beonly around 50-70%, effective in AIDS patients and to produce a muchhigher than usual incidence of adverse reactions (about 50%) (Wofsy, C.B. Antimicrobial Agents Annual, 1986, Vol 1, p377-400). There is thus aneed for new agents, especially for the prophylaxis of P.cariniipneumonia.

In another aspect the present invention provides the use of the compoundof formula (I) and physiologically acceptable salts thereof for themanufacture of a medicament for the treatment and/or prophylaxis ofPneumocystis carinii infections in mammals (including humans).

In a yet further aspect the present invention provides for the use ofthe compound of formula (I) and physiologically acceptable salts thereofto treat conditions resulting from relative or absolute T-celldeficiencies such as DiGeorge Syndrome, fungal infections, mycoplasmainfections, tuberculosis, leprosy, and systemic lupus erythemotosus.

In another aspect of the present invention provides for the use of thecompound of formula (I) and physiologically acceptable salts thereof forthe manufacture of a medicament for the treatment and/or prophylaxis ofcancer in mammals including humans).

In a further aspect of the present invention provides for the use of thecompound of formula (I) and physiologically acceptable salts thereof forthe manufacture of a medicament for the treatment and/or prophylaxis ofcancer other than radiotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a comparative graph showing concentration of antigen in vitrorelative to lymphocyte DNA synthesis for antigen with conventionaladjuvant alum and antigen+tucaresol; and

FIG. 2 is a comparative graph showing concentration of KLH relative to3H-thymidine incorporation for various administered concentrations oftucaresol including a control.

The compound of formula (I) or physiologically acceptable salts thereofcan also be used for the treatment and prophylaxis of cancer, at a dailydosage substantially lower and for a longer duration than the dosageused previously disclosed in EP 54924. The duration of dosing for thetreatment and prophylaxis of cancer using a compound of the invention islonger than would be required for radiosensitisation.

Examples of forms of cancers particularly suitable for treatment withthe compound of formula (I) are: melanoma, cervical cancer, breastcancer, colon cancer, cancer of the head and neck, gastric cancer, renalcancer, laryngeal cancer, rectal cancer, and non-Hodgkins lymphoma.Cancers that express tumour specific antigens or antigens rarelyexpressed or expressed at very low density on normal cells, are likelytherapeutic targets. Cancers which contain tumour specific cytotoxicT-cells which are anergic or otherwise ineffective are likely targetsfor therapy. Surgically resected tumours where there is a high risk ofrecurrence are also suitable for therapy with the compound of formula(I). Also early stage cancer patients with minimal disease or localiseddisease are suitable for therapy.

The compound of formula (I) is thought to act by providing aco-stimulatory signal to cloned (partially) activated T-cells in vitro,thus maximally activating T-cells.

A further aspect of the present invention provides for the use, as avaccine adjuvant, of the compound of formula (I) or physiologicallyacceptable salts. A vaccine may therefore be prepared by formulating theantigenic component with the compound or formula (I).

The compound or formula (I) may be administered to the human recipientby a route selected from oral, parenteral (including subcutaneous,intradermal, intramuscular and intravenous), rectal and inhalation. Thesize of an effective dose of a compound will depend upon a number offactors including the identity of the recipient, the type ofimmunopotentiation involved, the severity of the condition to be treatedand the route of administration, and will ultimately be at thediscretion of the attendant physician.

For each of the aforementioned conditions, such an effective dose willgenerally be in the range 0.5 to 50 mg/kg bodyweight of human recipientper day, preferably in the range 1 to 20 mg/kg bodyweight per day andmost preferably in the range 1 to 10 mg/kg bodyweight per day; anoptimum dose is 3 mg/kg bodyweight per day. The above doses are for ahuman usage.

The desired dose may be presented as between two and four sub-dosesadministered at appropriate intervals throughout the day. Thus wherethree sub-doses are employed each will generally lie in the range 0.03mg to 33 mg, preferably 16 mg to 166 mg and most preferably 0.3 to 6.6mg (acid)/kg bodyweight with an optimum of 1.0 mg (acid)/kg bodyweight.A daily dose for a human weighing of the order of 50 kg will thusgenerally lie in the range 5 mg to 5 g (acid), preferably in the range25 mg to 2.5 g (acid) and most preferably in the range 50 mg to 1 g(acid). Optimally a human daily dose is 150 mg (acid). The desired doseis preferably presented as a daily dose, over a period of at least 5days, most preferably over a period of at least 28 days.

While it is possible for the compound of formula (I) to be administeredas the raw chemical it is preferable to present them as a pharmaceuticalformulation preparation. The formulations of the present inventioncomprise a compound of formula (I), as above defined, together with oneor more acceptable carriers therefor and optionally other therapeuticingredients. The carrier(s) must be `acceptable` in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular and intravenous) and rectaladministration although the most suitable route may depend upon forexample the condition of the recipient. The formulations mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. All methods includethe step of bringing into association the compound of formula (I) (theactive ingredient) with the carrier which constitutes one or moreaccessory ingredients. In general the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both, and then,if necessary, shaping the product into the desired formulation.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression, or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, lubricating, surface active ordispersing agent. Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain antioxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Formulations for rectal administration may be presented as a suppositorywith the usual carriers such as cocoa butter.

European Patent No. 54924 contains no invitation to administer thecompound of formula (I) by the nasal or pulmonary route nor anysuggestion that the said compound, if administered in such a manner,would be effective in the treatment of the conditions therein taught;the said disclosure likewise contains no description of any formulationsuitable for administration by the nasal or pulmonary route.

Formulations suitable for pulmonary administration via the buccal cavityare presented such that particles containing the active ingredient anddesirably having a diameter in the range 0.5 to 7 microns are deliveredinto the bronchial tree of the recipient.

As one possibility such formulations are in the form of finelycomminuted powders which may conveniently be presented either in apierceable capsule, suitably of for example gelatin, for use in aninhalation device, or alternatively as a self-propelling formulationcomprising active ingredient, a suitable liquid propellant andoptionally other ingredients such as surfactant and/or a solid diluent.Self-propelling formulations may also be employed wherein the activeingredient is dispensed in the form of droplets of a solution orsuspension.

Such self-propelling formulations are analogous to those known in theart and may be prepared by established procedures. Suitably they arepresented in a container provided with either a mannually-operable orautomatically functioning valve having the desired spraycharacteristics; advantageously the valve is of a metered typedelivering a fixed volume, for example 50 to 100 microliters, upon eachoperation thereof.

As a further possibility the active ingredient may be in the form of asolution for use in an atomiser or nebuliser whereby an acceleratedairstream or ultrasonic agitation is employed to produce a fine dropletmist for inhalation.

Formulations suitable for nasal administration include presentationsgenerally similar to those described above for pulmonary administration.When dispensed such formulations should desirably have a particlediameter in the range 10 to 200 microns to enable retention in the nasalcavity; this may be achieved by, as appropriate, use of a powder of asuitable particle size or choice of an appropriate valve. Other suitableformulations include coarse powders having a particle diameter in therange 20 to 500 microns, for administration by rapid inhalation throughthe nasal passage from a container held close up to the nose, and nasaldrops comprising 0.2 to 5% w/v of the active ingredient in aqueous oroily solution.

Preferred unit dosage formulations are those containing an effectivedose, as hereinabove recited, or an appropriate fraction thereof, of theactive ingredient.

The following Examples are provided in illustration of the presentinvention and should not be construed as in any way constituting alimitation thereof. All temperatures are in degrees Celsius.

    ______________________________________    Solution for nebulisation    ______________________________________    Compound of formula (I)                           1.0    mg    Water for injections to                           10.0   mL    ______________________________________

Dissolve the compound of formula (I) as for injections. Sterilize thesolution by passage through a membrane filter, 0.21 m pore size,collecting the filtrate in a sterile receiver. Fill into sterile glassampoules, 10 mL/ampoule, under aseptic conditions and seal each ampouleby fusion of the glass.

    ______________________________________    Self-propelling formulation    ______________________________________    Compound of formula (I), micronised                             1.0 mg    Propellant to            5.0 mL    ______________________________________

Suspend the micronised compound of formula (I) in the propellant. Fillthis suspension under pressure into preformed, valved aerosol canisters,5 mL/canister, through the valve orifice.

The propellant is a commercially available mixture oftrichloromono-fluoromethane, dichlorodifluoromethane anddichlorotetrafluoroethane.

    ______________________________________    Powder for inhalation    ______________________________________    Compound of formula (I), micronised                           1.0 mg    Lactose               29.0 mg    ______________________________________

Triturate and blend the micronised compound of formula (I) with thelactose. Fill the resulting powder blend into hard gelatin capsuleshells, 30 mg per capsule. Alternatively, the micronised compound offormula (I) could be compressed into a plug and a device which deliverssmall amounts of the compound of formula (I) into the airstream can beused.

    ______________________________________    Nasal drops    ______________________________________    Compound of formula (I)                           100    mg    Methyl p-hydroxybenzoate                           10     mg    Water for injections to                           10     mL    ______________________________________

Dissolve the compound of formula (I) and the methyl p-hydroxybenzoate inthe water for injections. Fill this solution into suitable dropperbottles. 10 mL/bottle, and close by securing the dropper nozzle andbottle cap.

    ______________________________________    Tablet    ______________________________________    Compound of formula (I)                           100    mg    Lactose                100    mg    Starch                 50     mg    Polyvinylpyrrolidone   5      mg    Magnesium Stearate     5      mg                           260    mg    ______________________________________

The Compound, Lactose and Starch are mixed together and then granulatedwith a solution of Polyvinyl-pyrrolidone in water. After drying thegranules, the Magnesium Stearate is mixed in and tablets compressed atan average weight of 260 mg.

    ______________________________________    Capsule    ______________________________________    Compound of formula (I)                           100    mg    Dibasic Calcium Phosphate                           100    mg    Dihydrate    Sodium Starch Glycolate                           16     mg    Methylcellulose 400 cps                           5      mg    Stearic Acid           4      mg    Talc                   5      mg                           230    mg    ______________________________________

The Compound, Dibasic Calcium Phosphate, Dihydrate and Sodium StarchGlycolate are mixed together and then granulated with a solution of theMethylcellulose in water. After drying, the granules are mixed with theStearic Acid and Talc and the mixture filled into gelatin capsules at anaverage fill weight of 230 mg.

    ______________________________________    Suppository    ______________________________________    Compound of formula (I)                           100    mg    Suppository Base (Mixed                           1700   mg    Glycerides of saturated    fatty acids)                           1800   mg    ______________________________________

Grind the Compound to a particle size below 150μ. Add the suppositorybase at 38°-40° C. Mix to give a uniform dispersion. Pour intosuppository moulds and allow to cool.

    ______________________________________    Injection - Single dose intravenous    ______________________________________    Compound of formula (I) 100    mg    Sodium Hydroxide Solution (30%)                            q.s.    Water for Injections to 5      mL    ______________________________________

Suspend the Compound in some of the Water for Injections. Adjust the pHto 10 to 10.5 by addition of Sodium Hydroxide Solution. Add sufficientWater for Injections to produce the required final volume. Re-check thepH. Sterilise by passage through a sterile membrane filter of 0.22μ poresize. Fill under aseptic conditions into sterile vials and freeze dry.

    ______________________________________    Injection - Multidose, intramuscular    ______________________________________    Compound of formula (I), sterile                         1000   mg    Polysorbate 20       3      mg    Polyvinylpyrrolidone 1000   mg    Chlorocresol         60     mg    Sodium Chloride      q.s.   to isotonicity    Water for Injections to                         30     mL    ______________________________________

Dissolve the Polysorbate 20, Polyvinylpyrrolidone, Sodium Chloride andChlorocresol in Water for Injections. Sterile filter, 0.22μ. Grind thesterile Compound to a particle size below 20μ and add to the filteredsolution. Mix until a uniform dispersion is achieved. Fill into sterileglass vials.

    ______________________________________    Prolonged Release Tablet    ______________________________________    Compound of formula (I)                      200 mg    Casein            195 mg    Hydrogenated Castor Oil                      400 mg    Magnesium Stearate                       5 mg                      800 mg    ______________________________________

Melt the Hydrogenated Castor Oil and add the Compound, ground to aparticle size of less than 150μ. Add the Casein. Mix until uniform.Allow to cool and mill to a granule. Mix in the Magnesium Stearate andcompress to an average weight of 1,200 mg.

In the foregoing the "Compound" refers to a compound of formula (I) ashereinbefore defined.

Preferred unit dosage formulations are those containing a daily dose orunit daily sub-dose, as hereinabove recited, or an appropriate fractionthereof, of a compound of formula (I) or physiologically acceptablesalts thereof.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the an having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

The compound of formula (I) or physiologically acceptable salts thereofmay also be presented as depot formulations of the kind known in the artfrom which the active ingredient is released, over a prolonged period,once the formulation is in place within the body of the recipient.

The results of tests with the compound of formula (I) are described inthe following Experimental examples in order to illustrate the effect ofthe present invention in more detail.

EXAMPLE 1

Mice were injected subcutaneously in the dorsal mid-line either withantigen alone, with antigen plus a conventional adjuvant, or withantigen plus test compound. Subsequent additional injections of testcompound were also given where specified for up to four days at the sameinjection site. After seven days regional (inguinal) lymph nodes wereremoved and the lymph node cells restimulated with antigen alone.Antigen specific proliferation of lymphocytes was measured after afurther four days by a standard procedure employing ³ H-thymidine uptakeinto DNA and liquid scintillation spectrometry. Tests for humoralimmunity were performed by administering antigen and test compounds inthe same way. After 1-2 weeks blood was sampled by venepuncture andserum antibody was assayed by an enzyme-linked immunosorbent (ELISA)assay.

RESULTS

The data in FIG. 1 show the effects of the compound of formula (I) onT-lymphocyte priming to antigen (keyhole limper haemocyanin). BIOS micereceived 50 μg of antigen either alone (▪), with the conventionaladjuvant alum (⋄), or with 100 μg of compound of formula (I) (♦).Compound of formula (I) is also known as tucaresol.

EXAMPLE 2 Antitumour Activity of the Compound of Formula (I)

The activity of the compound of formula (I) was evaluated against theoutgrowth of subcutaneously implanted mouse colon adenocarcinoma 33(MCA38) in female C57BL/6 mice, using the following protocol:

Day 0 Forty, 7-week-old female C57BL/6 mice each implantedsubcutaneously with one 2 mm cube of tumour.

Day 12 19 mice each with palpable tumours (4 to 6 mm diameter) selectedfrom above group, randomized and assigned to either the test group (10animals) or the control group (9 animals).

Day 13 Test group each dosed with the compound of formula (I) (1 mg permouse dissolved in 0.2 ml PBS, intraperitoneally) once every alternateday (total of 7 doses),

Day 27 All mice killed, tumours excised and weighed. Mean tumour weightin test group compared with that in controls.

The compound of formula (I) was dissolved in PBS by dropwise addition of1M KOH to pH 10.0 followed by dropwise addition of conc. HCl to returnpH to neutral. Solutions were injected within 1 hour of preparation.

Dosing schedule where drug was given on alternate days was chosen inorder to extend the dosing period over 14 rather than 7 days. By sodoing tumours were exposed to the drug from day 13 right up until day 25(i.e. 2 days before the experiment was terminated). Results are shown inTable 1.

The pattern of tumour growth (diffuse granular) rendered callipermeasurements of volume, meaningless and such data are therefore notincluded.

EXAMPLE 3

Systemic (intraperitoneal) administration of tucaresol to produceimmunopotentiation.

Eight week old female Balb/c mice received five daily injections oftucaresol each containing either 100 μg, (□) 200 μg (♦) or 1 mg (⋄) ofthe drug intraperitoneally starting on day 0. A control group receivedno drug (▪). All mice were immunized subcutaneously on day 0 with 5 μgof KLH. An additional control group received no drug and no KLH (). Onday 1, concurrent with the second i.p. injection of drug mice wereimmunised subcutaneously in the dorsal mid-line at the base of the tailwith 10 μg of Keyhole limpet haemocyanin (KLH). On day 7 regional(inguinal) lymph nodes were removed and the lymph node cellsrestimulated with KLH in vitro. Proliferation was measured 4 days laterby the incorporation of ³ H-thymidine into DNA. This provided asensitive specific measure of the T-cell priming that has occurred invivo. The results are shown in FIG. 2. The results show that the dose oftucaresol required to produce immunopotentiation in this way is at leastfivefold lower than the dose required for left-shift effects onhaemoglobin and that the latter high doses are in fact much lesseffective in immunopotentiation than low doses.

Effect of tucaresol (4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid)treatment on SIV infection in cynomolgus macaques.

Four cynomolgus macaques (Macaca fascicularis) of average weight of 2.5kg with established SIV infection were used in this study. Two animalswere dosed with tucaresol at 30 mg/kg every other day for 9 days byintraperitoneal injection (5 injections total). The two untreatedanimals received control injections of saline. The treatment resulted insplenic enlargement in both treated animals, one of which also developeda general malaise which resolved over the next few days. Viral load wasmeasured on day eleven (two days after cessation of treatment). In theuntreated controls, 10³.5 viral units were detected per 10⁶ bloodleukocytes in both animals. In the treated animal who had no malaise10².5 viral units per 10⁶ leukocytes were detected (i.e. a tenfoldreduction in comparison with the two controls). In the treated animalwho had signs of malaise, no virus was detectable by the conventionalassay.

                  TABLE 1    ______________________________________                 TUMOR WEIGHT g.                              Untreated                 Compound of formula (I)                              Controls    ______________________________________                   1.44           1.66                   0.77           1.45                   0.72           1.43                   0.48           1.14                   0.43           0.56                   0.39           0.32                   0.28           0.26                   0.13           0.17                   0.06           Died                   0.03           --    MEAN WEIGHT g (SD)                   0.473(0.423)   0.868(0.50)    MEAN % REDUCTION              45.4    TUMOUR OUTGROWTH    TREATED v CONTROLS    MEDIAN WEIGHT  0.41           0.85    MEDIAN % REDUCTION            51.77    TUMOUR OUTGROWTH    TREATED v CONTROLS    ______________________________________     The compound of formula (I) inhibited the outgrowth of subcutaneously     implanted MCA38 in syngeneic C57BL/6 mice.

I claim:
 1. A method for treating virus infections in an immunodeficientmammal in need thereof, which comprises administering to said mammal aneffective immunopotentiatory amount of4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid or a physiologicallyacceptable salt thereof, over a period of at least 5 days for thepotentiation of an immune response.
 2. The method according to claim 1,wherein the immunodeficient mammal is human having a Stimulation Indexbelow
 14. 3. The method according to claim 1, wherein the effectiveamount is from 50 to 200 mg per day.
 4. The method according to claim 1,wherein the effective amount can be administered on alternate days. 5.The method according to claim 2, wherein the effective amount is from 50to 200 mg per day.
 6. The method according to claim 3, wherein theeffective amount can be administered on alternate days.
 7. A method ofreducing viral load of immunodeficiency viruses in the blood of a mammalhaving said viruses comprising administering to said mammal an effectiveviral load reducing amount of 4-(2-formyl-3-hydroxyphenoxymethyl)benzoicacid or a physiologically acceptable salt thereof.